Extracts, compounds and pharmaceutical compositions having anti-diabetic activity and their use

ABSTRACT

The invention relates inter alia to pharmaceutical compositions containing an extract obtainable from a plant of the genus  Trichocaulon  or  Hoodia  having anti-diabetic activity; and to the use of such extracts and to compound (1) as herein defined and its analogues for the manufacture of medicaments having anti-diabetic activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority 35 U.S.C 119 to U.K.Application No. 0016213.1 filed Jun. 30, 2000 and is incorporated hereinin its entirety by reference.

1. Field of the Invention

This invention relates to a new use for steroidal glycosides andcompositions containing them for use in the prevention and treatment ofdiabetes.

In a particular application, the invention relates to an anti-diabeticagent, to an anti-diabetic composition containing the anti-diabeticagent, and to a method for treating diabetes.

2. Description of the Related Art

The International application WO 98/46243 discloses steroidal glycosideshaving appetite suppressant activity. In particular, it describesextracts from the genus Trichocaulon or of the genus Hoodia and havingappetite suppressant activity; these extracts include the compound ofgeneral formula (1):

In accordance with S.I. nomenclature, the active principle (1) is thecompound3-0-[-β-D-thevetopyranosyl-(1-4)-β-D-cymaropyranosyl-(1-4)-β-D-cymaropyranosyl]-12β-0-tigloyloxy-14-hydroxy-14β-pregn-50-en-20-one(C₄₇H₇₄O₁₅ M⁺878).

Also, WO 98/46243 discloses further active analogues or steroidalglycosides derivatives of general formula (2), (3), (4), (5), (6), (7),(8), (9), (10), (11), (12), (13), (14) (see herein below) havingappetite suppressant activity.

Diabetes is a deficiency condition marked by a habitual discharge of anexcessive quantity of urine; in particular, it includes diabetesmellitus, which is a metabolic disorder in which the ability to oxidizecarbohydrates is more or less completely lost, usually due to faultypancreatic activity, especially of the islets of Langerhans, andconsequent disturbance of normal insulin mechanism. This produceshyperglycemia with resulting glycosuria and polyuria giving symptoms ofthirst, hunger, emaciation and weakness and also imperfect combustion offats with resulting acidosis, sometimes leading to dyspnea, lipemia,ketonuria, and finally coma; there may also be pruritus and loweredresistance to pyogenic infections (Dorland's Medical Dictionary—24^(th)Edition—W.B.Saunders Company).

The diabetic disease state is characterized by an impaired glucosemetabolism that manifests itself in, inter alia, elevated glucose levelsin patients suffering therefrom. Generally, diabetes is classified intotwo distinct subgroups:

(1) Type 1 diabetes, or insulin-dependent diabetes mellitus (IDDM),which arises when patients lack β-cells producing insulin in theirpancreatic glands, and

(2) Type 2 diabetes, or non-insulin dependent diabetes mellitus (NIDDM),which occurs in patients with, inter alia, impaired β-cell function.

At present, Type 1 diabetic patients are treated with insulin, while themajority of Type 2 diabetic patients are treated with hypoglycemicagents, such as sulfonylureas that stimulate β-cell function, with otheragents that enhance the tissue selectivity of the patients towardsinsulin, or with insulin itself. Unfortunately, the use of insulincurrently requires multiple daily doses, normally administered byself-injection, with determination of the proper dosage of insulinrequiring frequent estimations of the sugar in urine or blood, performedeither by the patient or the administering physician. The unintendedadministration of an excess dose of insulin can result in hypoglycemia,with adverse effects ranging from mild abnormalities in blood glucose tocoma, or even death. Although hypoglycemic agents such as sulfonylureashave been employed widely in the treatment of NIDDM, this treatment is,in many instances, not completely satisfactory. Where existingtreatments prove ineffective in normalizing blood sugar levels ofpatients, there is an increased risk of acquiring diabeticcomplications. Also, many patients gradually lose the ability to respondto treatment with sulfonylureas and are thus gradually forced intoinsulin treatment. Since many extant forms of diabetic therapy haveproven ineffective achieving satisfactory glycemic control, therecontinues to be a great demand for novel therapeutic approaches.

SUMMARY OF THE INVENTION

According to the invention, it has been found that the extracts from aplant of the genus Trichocaulon or of the genus Hoodia, the compound ofgeneral formula (1), as well as the steroidal glycosides derivatives ofgeneral formula (2), (3), (4), (5), (6), (7), (8), (9), (10), (11),(12), (13), (14) (see herein below) have anti-diabetic activity.

The present invention is particularly concerned with the treatment ofType II diabetes and the corresponding anti-diabetic agents. Theinvention provides a new use for steroidal glycosides and compositionscontaining them for use in the prevention and treatment of diabetes. Theinvention provides an anti-diabetic agent, an anti-diabetic compositioncontaining the anti-diabetic agent, a foodstuff or beverage containingthe anti-diabetic agent, kits based on the anti-diabetic agent, a methodfor preventing or treating diabetes or impaired glucose tolerance, and amethod of decreasing blood glucose level. The anti-diabetic agent may bean extract from a plant of the genus Trichocaulon or of the genusHoodia, a compound of general formula (1), as well as the steroidalglycosides derivatives of general formula (2), (3), (4), (5), (6), (7),(8), (9), (10), (11), (12), (13), or (14), each as described hereinbelow.

DETAILED DESCRIPTION OF THE INVENTION Definitions

“Acyl” means an H—CO— or Alkyl-CO— group wherein the alkyl group is asherein described. Preferred acyls contain a lower alkyl. Exemplary acylgroups include formyl, acetyl, propanoyl, 2-methylpropanoyl, butanoyland palmitoyl.

“Alkyl” means an aliphatic hydrocarbon group which may be straight orbranched having about 1 to about 20 carbon atoms in the chain. Preferredalkyl groups have 1 to about 12 carbon atoms in the chain. Branchedmeans that one or more lower alkyl groups such as methyl, ethyl orpropyl are attached to a linear alkyl chain. “Lower alkyl” means about 1to about 4 carbon atoms in the chain which may be straight or branched.Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl,n-butyl, t-butyl, n-pentyl, 3-pentyl.

“Aryl” means an aromatic monocyclic or multicyclic ring system of about6 to about 14 carbon atoms, preferably of about 6 to about 10 carbonatoms. The aryl is optionally substituted with one or more ring systemsubstituents which may be the same or different, and are as definedherein. Exemplary aryl groups include phenyl or naphthyl, or phenylsubstituted or naphthyl substituted.

The term “pharmaceutical composition” means a composition comprising acompound of general formula (1), (2), (3), (4), (5), (6), (7), (8), (9),(10), (11), (12), (13), (14), or an extract in accordance with thisinvention, and at least one component selected from the group comprisingpharmaceutically acceptable carriers, diluents, adjuvants, excipients,or vehicles, such as preserving agents, fillers, disintegrating agents,wetting agents, emulsifying agents, suspending agents, sweeteningagents, flavoring agents, perfuming agents, antibacterial agents,antifungal agents, lubricating agents and dispensing agents, dependingon the nature of the mode of administration and dosage forms.

“Pharmaceutically acceptable” means it is, within the scope of soundmedical judgement, suitable for use in contact with the cells of humansand animals without undue toxicity, irritation, allergic response andthe like, and are commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable dosage forms” means dosage forms of thecompound of the invention, and includes, for example, tablets, dragees,powders, elixirs, syrups, liquid preparations, including suspensions,sprays, inhalants tablets, lozenges, emulsions, solutions, granules,capsules and suppositories, as well as liquid preparations forinjections, including liposome preparations. Techniques and formulationsgenerally may be found in Remington, Pharmaceutical Sciences, MackPublishing Co., Easton, Pa., latest edition.

“Pharmaceutically acceptable prodrugs” as used herein means thoseprodrugs of the compounds useful according to the present inventionwhich are, within the scope of sound medical judgment, suitable for usein contact with the tissues of humans and animals with undue toxicity,irritation, allergic response, and the like, commensurate with areasonable benefit/risk ratio, and effective for their intended use, aswell as the zwitterionic forms, where possible, of the compounds of theinvention. The term “prodrug” means compounds that are rapidlytransformed in vivo to yield the parent compound of the above formula,for example by hydrolysis in blood. Functional groups which may berapidly transformed, by metabolic cleavage, in vivo form a class ofgroups reactive with the carboxyl group of the compounds of thisinvention. Because of the ease with which the metabolically cleavablegroups of the compounds useful according to this invention are cleavedin vivo, the compounds bearing such groups act as pro-drugs. A thoroughdiscussion of prodrugs is provided in the following: Design of Prodrugs,H. Bundgaard, ed., Elsevier, 1985; Methods in Enzymology, K. Widder etal, Ed., Academic Press, 42, p. 309-396, 1985; A Textbook of Drug Designand Development, Krogsgaard-Larsen and H. Bundgaard, ed., Chapter 5;Design and Applications of Prodrugs p. 113-191, 1991; Advanced DrugDelivery Reviews, H. Bundgard, 8, p. 1-38, 1992; Journal ofPharmaceutical Sciences, 77, p. 285, 1988; Chem. Pharm. Bull., N. Nakeyaet al, 32, p. 692, 1984; Pro-drugs as Novel Delivery Systems, T. Higuchiand V. Stella, Vol. 14 of the A.C.S. Symposium Series, and BioreversibleCarriers in Drug Design, Edward B. Roche, ed., American PharmaceuticalAssociation and Pergamon Press, 1987, which are incorporated herein byreference.

“Pharmaceutically acceptable salts” means the relatively non-toxic,inorganic and organic acid addition salts, and base addition salts, ofcompounds of the present invention. These salts can be prepared in situduring the final isolation and purification of the compounds. Inparticular, acid addition salts can be prepared by separately reactingthe purified compound in its free base form with a suitable organic orinorganic acid and isolating the salt thus formed. See, for example S.M. Berge, et al., Pharmaceutical Salts, J. Pharm. Sci., 66: p. 1-19(1977) which is incorporated herein by reference. Base addition saltscan also be prepared by separately reacting the purified compound in itsacid form with a suitable organic or inorganic base and isolating thesalt thus formed. Base addition salts include pharmaceuticallyacceptable metal and amine salts.

The term “animal” as used herein extends to, but is not restricted to,companion animals, e.g. household pets and domesticated animals;non-limiting examples of such animals include cattle, sheep, ferrets,swine, camels, horses, poultry, fish, rabbits, goats, dogs and cats.

According to the present invention, and as hereinbefore and hereaftermentioned:

-   “diabetes” preferably refers to non-insulin dependent diabetes (type    II);-   “anti-diabetic” means the activity useful for the “treatment” of    “diabetes”,    which includes the prevention of the development of diabetes, and/or    the treatment of established diabetes; it also includes the    prevention of the causes of diabetes, and/or the decrease or    disappearance of its symptoms and/or consequences.

In particular, it has been found that compounds of the invention have atleast the following double therapeutic effect:

-   1) the prevention of diabetes, since the compounds of the invention    can treat impaired glucose tolerance;-   2) the actual treatment of established diabetes since the compounds    of the invention can decrease the blood glucose level.

According to a first embodiment, the invention concerns the use of anextract from a plant of the genus Trichocaulon or Hoodia, as describedin WO 98/46243 (the contents of which are incorporated herein byreference thereto) in the manufacture of a medicament havinganti-diabetic activity.

Preferably, the said extract comprises as an active ingredient acompound of general formula (1):

and/or a pharmaceutically acceptable salt or prodrug thereof.

According to a further aspect, the invention also concerns the saidextract for use as a medicament having anti-diabetic activity.

The invention also extends to a pharmaceutical composition havinganti-diabetic activity comprising an effective quantity of the saidextract; and to compounds of formula (1) having anti-diabetic activity.

It is also provided a method for treating diabetes by administering to ahuman or animal an effective dosage of the said extract or the saidcomposition.

According to a still further aspect, the invention also concerns the useof the said extract in the manufacture of a foodstuff or beverage tohave an anti-diabetic effect when ingested.

The said foodstuff or beverage comprising an effective quantity of thesaid extract to have an anti-diabetic effect when ingested is also partof the present invention.

According to a further embodiment, the invention concerns the use of oneor more steroidal glycosides derivatives of general formula (A) (seebelow) and their pharmaceutically acceptable salts and pro-drugs in themanufacture of a medicament having anti-diabetic activity:

In the general formula (A):

R=alkyl;

R₁=H, alkyl, tigloyl, anthraniloyl, or any other organic ester group;

R₂=H, or one or more 6-deoxy carbohydrates, or one or more 2,6-dideoxycarbohydrates, or glucose molecules, or combinations thereof;

R₃=H, alkyl, aryl, acyl, or glucoxy,

R₄, R₅=either R₄, R₅ form together with the Carbon atom which they areattached to a carbonyl group (—C═O), or R₄=H and R₅=H, OH;

R₆, R₇=either R₆, R₇ form together with the Carbon atom C-16 which theyare attached to a carbonyl group (—C═O), or R₆=H and R₇=—OR₃;

R₈=H, OH;

and

the broken lines indicate the optional presence of a further bondbetween C4-C5 or C5-C6, and/or C14-C15.

Preferably, compounds of general formula (A) can be chosen from thefollowing families of formula (2), (3), (4), (5), (6), (7), (8), (9),(10), (11), (12), (13) or (14) below, as described in WO 98/46243 andincorporated herein by reference:

and their pharmaceutically acceptable salts and pro-drugs.

In the general formula (2), (3), (4), (5), (6), (7), (8), (9), (10),(11), (12), (13) above: R, R₁, R₂, R₃ are as defined above;

and in the general formula (14): R=H, alkyl, aryl or any steroidpossessing a C14 beta hydroxy group, or a C12 beta hydroxyfunctionality, or a C17 acyl group, or a C5- C6 olefin, or combinationsthereof, as described in WO 98/46243.

According to a still more preferred aspect, compounds of general formula(A) are represented by formula (1):

and its pharmaceutically acceptable salts and pro-drugs.

According to a preferred embodiment, the invention concerns the use ofthe compound of general formula (1) (see herein above) as described inWO 98/46243 and incorporated herein by reference in the manufacture of amedicament having anti-diabetic activity.

According to a further aspect, the invention also concerns the saidcompounds of general formula (A), including that of formula (1) (seeherein above) for use as a medicament having anti-diabetic activity.

The invention also extends to a pharmaceutical composition havinganti-diabetic activity comprising an effective quantity of one or moreof the said compounds of general formula (A), preferably that of formula(1) (see herein above). It is also provided a method for treatingdiabetes by administering to a human or animal an effective dosage ofone or more of the said derivatives of general formula (A), preferablythat of formula (1) (see herein above) or the said compositions.

According to a still further aspect, the invention also concerns the useof one or more of the said derivatives of general formula (A),preferably that of formula (1), (see herein above) in the manufacture ofa foodstuff or beverage to have an anti-diabetic effect when ingested.

The said foodstuff or beverage comprising an effective quantity of oneor more of the said derivatives of general formula (A), preferably thatof formula (1) (see herein above) to have an anti-diabetic effect wheningested, is also part of the present invention.

As described in WO 98/46243 and incorporated herein by reference, theactive ingredient may be an extract from a plant of the genusTrichocaulon or Hoodia, or a compound of the formula (1) (eitherextracted from a plant of the genus Trichocaulon or Hoodia or preparedsynthetically) or a derivative thereof.

The plant may be of the species Trichocaulon officinale or Trichocaulonpiliferum, or the species Hoodia currorii, Hoodia gordonii or Hoodialugardii.

Preferably, the compounds of the invention are prepared inpharmaceutically acceptable dosage forms. The anti-diabetic compositionor formulation may consist of the anti-diabetic agent admixed with apharmaceutical excipient, diluent or carrier. Other suitable additives,including a stabilizer and such other ingredients as may be desired maybe added.

The composition may be prepared in unit dosage form.

As an anti-diabetic agent, a compound of formula (A), preferably offormula (1), or the composition as herein above mentioned, isadvantageously administered to said human in a dosage amount of fromabout 0.05 mg/kg/day to about 100 mg/kg/day. A preferred dosage range isabout 0.1 mg/kg/day to about 50 mg/kg/day. When using the spray driedpowder form of the extract of this invention, a preferred dosage rangeis about 0.5 mg/kg/day to about 100 mg/kg/day; especially preferred isabout 1 mg/kg/day to about 50 mg/kg/day.

According to a further aspect, the invention also concerns apharmaceutical composition comprising an effective amount of:

-   i) an extract as mentioned above or a compound of formula (A), (1),    (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13)    or (14) as described above,    in association with-   ii) one or more other agents chosen from: representative agents to    treat diabetes, glycogen phosphorylase inhibitors, sorbitol    dehydrogenase inhibitors, glucosidase inhibitors, aldose reductase    inhibitors;

Representative agents that can be used to treat diabetes include insulinand insulin analogs: (e.g., LysPro insulin, inhaled formulationscomprising insulin); GLP-1 (7-37) (insulinotropin) and GLP-1 (7-36)-NH₂;sulfonylureas and analogs: chlorpropamide, glibenclamide, tolbutamide,tolazamide, acetohexamide, glypizide, glimepiride, repaglinide,meglitinide; biguanides: metformin, phenformin, buformin; a2-antagonistsand imidazolines: midaglizole, isaglidole, deriglidole, idazoxan,efaroxan, fluparoxan; other insulin secretagogues: linogliride,insulinotropin, exendin-4, BTS-67582, A-4166; glitazones: ciglitazone,pioglitazone, englitazone, troglitazone, darglitazone, rosiglitazone;PPAR-gamma agonists; RXR agonists: JTT-501, MCC-555, MX-6054,DRF2593,GI-262570, KRP-297, LG100268; fatty acid oxidation inhibitors:clomoxir, etomoxir; α-glucosidase inhibitors: precose, acarbose,miglitol, emiglitate, voglibose, MDL-25,637, camiglibose, MDL-73,945;β-agonists: BRL 35135, BRL 37344, Ro 16-8714, ICI D7114, CL 316,243,TAK-667, AZ40140; phosphodiesterase inhibitors, both cAMP and cGMP type:sildenafil, L686398: L-386,398; lipid-lowering agents: benfluorex,atorvastatin; antiobesity agents: fenfluramine, orlistat, sibutramine;vanadate and vanadium complexes (e.g., Naglivan®) and peroxovanadiumcomplexes; amylin antagonists: pramlintide, AC-137; lipoxygenaseinhibitors: masoprocal; somatostatin analogs: BM-23014, seglitide,octreotide; glucagon antagonists: BAY 276-9955; insulin signalingagonists, insulin mimetics, PTP1B inhibitors: L-783281, TER17411,TER17529; gluconeogenesis inhibitors: GP3034; somatostatin analogs andantagonists; antilipolytic agents: nicotinic acid, acipimox, WAG 994;glucose transport stimulating agents: BM-130795; glycogen phosphorylaseinhibitors: glucose synthase kinase inhibitors: lithium chloride,CT98014, CT98023; galanin receptor agonists; MTP inhibitors such asthose disclosed in U.S. provisional patent application No. 60/164,803;growth hormone secretagogues such as those disclosed in PCT publicationnumbers WO 97/24369 and WO 98/58947; NPY antagonists: PD-160170, BW-383,BW1229, CGP-71683A, NGD 95-1, L-152804; anorectic agents inlcuding 5-HTand 5-HT2C receptor antagonists and/or mimetics: dexfenfluramine,Prozac®, Zoloft®; CCK receptor agonists: SR-27897B; galanin receptorantagonists; MCR-4 antagonists: HP-228; leptin or mimetics: leptin;11-beta-hydroxysteroid dehydrogenase type-I inhibitors; urocortinmimetics, CRF antagonists, and CRF binding proteins: RU-486, urocortin.Other anti-diabetic agents that can be used include ergoset andD-chiroinositol. Other anti-diabetic agents will be known to thoseskilled in the art.

Any glycogen phosphorylase inhibitor may be used as the second compoundof this invention. The term glycogen phosphorylase inhibitor refers toany substance or agent or any combination of substances and/or agentswhich reduces, retards, or eliminates the enzymatic action of glycogenphosphorylase. The currently known enzymatic action of glycogenphosphorylase is the degradation of glycogen by catalysis of thereversible reaction of a glycogen macromolecule and inorganic phosphateto glucose-1-phosphate and a glycogen macromolecule which is oneglucosyl residue shorter than the original glycogen macromolecule(forward direction of glycogenolysis). Such actions are readilydetermined by those skilled in the art according to standard assays(e.g., as described hereinafter). A variety of these compounds areincluded in the following published PCT patent applications: PCTapplication publication WO 96/39384 and WO96/39385. However, otherglycogen phosphorylase inhibitors will be known to those skilled in theart.

Any sorbitol dehydrogenase inhibitor may be used as the second compoundof the invention. Sorbitol dehydrogenase inhibitors lower fructoselevels and have been used to treat or prevent diabetic complicationssuch as neuropathy, retinopathy, nephropathy, cardiomyopathy,microangiopathy, and macroangiopathy. U.S. Pat. Nos. 5,728,704 and5,866,578 disclose compounds and a method for treating or preventingdiabetic complications by inhibiting the enzyme sorbitol dehydrogenase.

A glucosidase inhibitor inhibits the enzymatic hydrolysis of complexcarbohydrates by glycoside hydrolases, for example amylase or maltase,into bioavailable simple sugars, for example, glucose. The rapidmetabolic action of glucosidases, particularly following the intake ofhigh levels of carbohydrates, results in a state of alimentaryhyperglycemia which, in adipose or diabetic subjects, leads to enhancedsecretion of insulin, increased fat synthesis and a reduction in fatdegradation. Following such hyperglycemias, hypoglycemia frequentlyoccurs, due to the augmented levels of insulin present. Additionally, itis known that both hypoglycemias and chyme remaining in the stomachpromotes the production of gastric juice, which initiates or favors thedevelopment of gastritis or duodenal ulcers. Accordingly, glucosidaseinhibitors are known to have utility in accelerating the passage ofcarbohydrates through the stomach and inhibiting the absorption ofglucose from the intestine. Furthermore, the conversion of carbohydratesinto lipids of the fatty tissue and the subsequent incorporation ofalimentary fat into fatty tissue deposits is accordingly reduced ordelayed, with the concomitant benefit of reducing or preventing thedeleterious abnormalities resulting therefrom.

Any glucosidase inhibitor may be employed in combination with theextracts of this invention and with the compounds of Formula (A), thestereoisomers and prodrugs thereof, and the pharmaceutically acceptablesalts of the compounds, stereoisomers, and prodrugs; however, generallypreferred glucosidase inhibitors comprise amylase inhibitors. An amylaseinhibitor is a glucosidase inhibitor that inhibits the enzymaticdegradation of starch or glycogen into maltose. The inhibition of suchenzymatic degradation is beneficial in reducing amounts of bioavailablesugars, including glucose and maltose, and the concomitant deleteriousconditions resulting therefrom.

A variety of glucosidase inhibitors will be known to one of ordinaryskill in the art. However, in the practice of the pharmaceuticalcompositions, combinations, methods, and kits of the instant invention,generally preferred glucosidase inhibitors are those inhibitors selectedfrom the group consisting of acarbose, adiposine, voglibose, miglitol,emiglitate, MDL-25637, camiglibose, tendamistate, AI-3688, trestatin,pradimicin-Q and salbostatin.

The glucosidase inhibitor acarbose,O-4,6-dideoxy-4-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)-2-cyclohexen-1-yl]amino]-α-glucopyranosyl-(1--->4)-O-α-D-glucopyranosyl-(1 --->4)-D-glucose, the various amino sugarderivatives related thereto and a process for the preparation thereof bythe microbial cultivation of Actinoplanes strains SE 50 (CBS 961.70), SB18 (CBS 957.70), SE 82 (CBS 615.71), SE 50/13 (614.71) and SE 50/110(674.73) are disclosed in U.S. Pat. Nos. 4,062,950 and 4,174,439respectively.

The glucosidase inhibitor adiposine, consisting of adiposine forms 1 and2, is disclosed in U.S. Pat. No. 4,254,256. Additionally, a process forthe preparation and purification of adiposine is disclosed in Namiki etal., J. Antiobiotics, 35, 1234-1236 (1982). The glucosidase inhibitorvoglibose,3,4-dideoxy-4-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-2-C-(hydroxymethyl)-D-epi-inositol,and the various N-substituted pseudo-aminosugars related thereto, aredisclosed in U.S. Pat. No. 4,701,559.

The glucosidase inhibitor miglitol,(2R,3R,4R,5S)-1-(2-hydroxyethyl)-2-(hydroxymethyl)-3,4,5-piperidinetriol,and the various 3,4,5-trihydroxypiperidines related thereto, aredisclosed in U.S. Pat. No. 4,639,436.

The glucosidase inhibitor emiglitate, ethylp-[2-[(2R,3R,4R,5S)-3,4,5-trihydroxy-2-(hydroxymethyl)piperidino]ethoxy]-benzoate,the various derivatives related thereto and pharmaceutically acceptableacid addition salts thereof, are disclosed in U.S. Pat. No. 5,192,772.

The glucosidase inhibitor MDL-25637,2,6-dideoxy-7-O-β-D-glucopyrano-syl-2,6-imino-D-glycero-L-gluco-heptitol,the various homodisaccharides related thereto and the pharmaceuticallyacceptable acid addition salts thereof, are disclosed in U.S. Pat. No.4,634,765.

The glucosidase inhibitor camiglibose, methyl6-deoxy-6-[(2R,3R,4R,5S)-3,4,5-trihydroxy-2-(hydroxymethyl)piperidino]-β-D-glucopyranosidesesquihydrate, the deoxy-nojirimycin derivatives related thereto, thevarious pharmaceutically acceptable salts thereof and synthetic methodsfor the preparation thereof, are disclosed in U.S. Pat. Nos. 5,157,116and 5,504,078.

The glucosidase inhibitor pradimicin-Q and a process for the preparationthereof by the microbial cultivation of Actinomadura verrucosporastrains R103-3 or A10102, are disclosed in U.S. Pat. Nos. 5,091,418 and5,217,877 respectively.

The glycosidase inhibitor salbostatin, the various pseudosaccharidesrelated thereto, the various pharmaceutically acceptable salts thereofand a process for the preparation thereof by the microbial cultivationof Streptomyces albus strain ATCC 21838, are disclosed in U.S. Pat. No.5,091,524.

Any aldose reductase inhibitor may be used in the pharmaceuticalcompositions, methods and kits of this invention. The term aldosereductase inhibitor refers to a compound which inhibits thebioconversion of glucose to sorbitol catalyzed by the enzyme aldosereductase. Such inhibition is readily determined by those skilled in theart according to standard assays (J. Malone, Diabetes, 29:861-864, 1980.“Red Cell Sorbitol, an Indicator of Diabetic Control”). The followingpatents and patent applications, each of which is hereby whollyincorporated herein by reference, exemplify aldose reductase inhibitorswhich can be used in the compositions, methods and kits of thisinvention, and refer to methods of preparing those aldose reductaseinhibitors: U.S. Pat. No. 4,251,528; U.S. Pat. No. 4,600,724; U.S. Pat.4,464,382, U.S. Pat. No. 4,791,126, U.S. Pat. No. 4,831,045; U.S. Pat.No. 4,734,419; U.S. Pat. No. 4,883,800; U.S. Pat. No. 4,883,410; U.S.Pat. No. 4,883,410; U.S. Pat. No. 4,771,050; U.S. 5,252,572; U.S. Pat.No. 5,270,342; U.S. Pat. No. 5,430,060; U.S. Pat. No. 4,130,714; U.S.Pat. No. 4,540,704; U.S. Pat. No. 4,438,272; U.S. Pat. No. 4,436,745,U.S. Pat. No. 4,438,272; U.S. Pat. No. 4,436,745, U.S. Pat. No.4,438,272; U.S. Pat. No. 4,436,745, U.S. Pat. No. 4,438,272; U.S. Pat.No. 4,980,357; U.S. Pat. No. 5,066,659; U.S. Pat. No. 5,447,946; U.S.Pat. No. 5,037,831.

A variety of aldose reductase inhibitors are specifically described andreferenced below, however, other aldose reductase inhibitors will beknown to those skilled in the art. Also, common chemical USAN names orother designations are in parentheses where applicable, together withreference to appropriate patent literature disclosing the compound.

Accordingly, examples of aldose reductase inhibitors useful in thecompositions, methods and kits of this invention include:

-   1. 3-(4-bromo-2-fluorobenzyl)-3,4-dihydro-4-oxo-1-phthalazineacetic    acid (ponalrestat, U.S. Pat. No. 4,251,528);-   2.    N[[(5-trifluoromethyl)-6-methoxy-1-naphthalenyl]thioxomethyl}-N-methylglycine    (tolrestat, U.S. Pat. No. 4,600,724);-   3. 5    -[(Z,E)-βP-methylcinnamylidene]-4-oxo-2-thioxo-3-thiazolideneacetic    acid (epalrestat, U.S. Pat. No. 4,464,382, U.S. Pat. No. 4,791,126,    U.S. Pat. No. 4,831,045);-   4. 3-(4-bromo-2-fluorobenzyl)-7-chloro-3,4-dihydro-2,4-dioxo-1    (2H)-quinazolineacetic acid (zenarestat, U.S. Pat. No. 4,734,419,    and U.S. Pat. No. 4,883,800);-   5. 2R,4R-6,7-dichloro-4-hydroxy-2-methylchroman-4-acetic acid (U.S.    Pat. No. 4,883,410);-   6. 2R,4R-6,7-dichloro-6-fluoro-4-hydroxy-2-methylchroman-4-acetic    acid (U.S. Pat. No. 4,883,410);-   7. 3,4-dihydro-2,8-diisopropyl-3-oxo-2H-1,4-benzoxazine-4-acetic    acid (U.S. Pat. No. 4,771,050);-   8.    3,4-dihydro-3-oxo-4-[(4,5,7-trifluoro-2-benzothiazolyl)methyl]-2H-1,4-benzothiazine-2-acetic    acid (SPR-210, U.S. Pat. No. 5,252,572);-   9.    N-[3,5-dimethyl-4-[(nitromethyl)sulfonyl]phenyl]-2-methyl-benzeneacetamide    (ZD5522, U.S. Pat. No. 5,270,342 and U.S. Pat. No. 5,430,060);-   10. (S)-6-fluorospiro[chroman-4,4′-imidazolidine]-2,5¢-dione    (sorbinil, U.S. Pat. No. 4,130,714);-   11.    d-2-methyl-6-fluoro-spiro(chroman-4′,4′-imidazolidine)-2′,5′-dione    (U.S. Pat. No. 4,540,704);-   12. 2-fluoro-spiro(9H-fluorene-9,4′-imidazolidine)-2′,5′-dione (U.S.    Pat. No. 4,438,272);-   13. 2,7-di-fluoro-spiro(9H-fluorene-9,4′-imidazolidine)-2′,5′-dione    (U.S. Pat. No. 4,436,745, U.S. Pat. No. 4,438,272);-   14.    2,7-di-fluoro-5-methoxy-spiro(9H-fluorene-9,4′-imidazolidine)-2′,5′-dione    (U.S. Pat. No. 4,436,745, U.S. Pat. No. 4,438,272);-   15.    7-fluoro-spiro(5H-indenol[1,2-b]pyridine-5,3′-pyrrolidine)-2,5′-dione    (U.S. Pat. No. 4,436,745, U.S. Pat. No. 4,438,272);-   16.    d-cis-6′-chloro-2′,3′-dihydro-2′-methyl-spiro-(imidazolidine-4,4′-4′H-pyrano(2,3-b)pyridine)-2,5-dione    (U.S. Pat. No. 4,980,357);-   17. spiro[imidazolidine-4,5′ (6H)-quinoline]-2,5-dione-3′-chloro-7′,    8′-dihydro-7′-methyl-(5′-cis) (U.S. Pat. No. 5,066,659);-   18.    (2S,4S)-6-fluoro-2′,5′-dioxospiro(chroman-4,4′-imidazolidine)-2-carboxamide    (fidarestat, U.S. Pat. No. 5,447,946); and-   19.    2-[(4-bromo-2-fluorophenyl)methyl]-6-fluorospiro[isoquinoline-4(1H),3′-pyrrolidine]-1,2′,3,5′    (2H)-tetrone (minalrestat, U.S. Pat. No. 5,037,831).

Other aldose reductase inhibitors include compounds of formula (B):

and pharmaceutically acceptable salts thereof, wherein

Z in the compound of formula B is O or S;

R¹ in the compound of formula B is hydroxy or a group capable of beingremoved in vivo to produce a compound of formula B wherein R¹ is OH; and

X and Y in the compound of formula B are the same or different and areselected from hydrogen, trifluoromethyl, fluoro, and chloro.

A preferred subgroup within the above group of aldose reductaseinhibitors includes numbered compounds 1, 2, 3, 4, 5, 6, 9, 10, and 17,and the following compounds of formula B:

-   20.    3,4-dihydro-3-(5-fluorobenzothiazol-2-ylmethyl)-4-oxophthalazin-1-yl-acetic    acid [R¹=hydroxy; X=F; Y=H];-   21.    3-(5,7-difluorobenzothiazol-2-ylmethyl)-3,4-dihydro-4-oxophthalazin    1-ylacetic acid [R¹=hydroxy; X=Y=F];-   22. 3-(5-chlorobenzothiazol-2-ylmethyl)-3,4-dihydro-4-oxophthalazin-    1-ylacetic acid [R¹=hydroxy; X=Cl; Y=H];-   23.3    -(5,7-dichlorobenzothiazol-2-ylmethyl)-3,4-dihydro-4-oxophthalazin-1-ylacetic    acid [R¹=hydroxy; X═Y═Cl];-   24.    3,4-dihydro-4-oxo-3-(5-trifluoromethylbenzoxazol-2-ylmethyl)phthalazin-1-ylacetic    acid [R¹=hydroxy; X═CF₃; Y═H];-   25.    3,4-dihydro-3-(5-fluorobenzoxazol-2-ylmethyl)-4-oxophthalazin-1-yl-acetic    acid [R¹=hydroxy; X═F; Y═H];-   26.    3-(5,7-difluorobenzoxazol-2-ylmethyl)-3,4-dihydro-4-oxophthalazin-1-ylacetic    acid [R¹=hydroxy; X═Y═F];-   27.    3-(5-chlorobenzoxazol-2-ylmethyl)-3,4-dihydro-4-oxophthalazin-1-ylacetic    acid [R¹=hydroxy; X═Cl; Y═H];-   28.    3-(5,7-dichlorobenzoxazol-2-ylmethyl)-3,4-dihydro-4-oxophthalazin-    1-ylacetic acid [R¹=hydroxy; X═Y═Cl]; and-   29. zopolrestat; 1-phthalazineacetic acid,    3,4-dihydro-4-oxo-3-[[5-(trifluoromethyl)-2-benzothiazolyl]methyl]-[R¹=hydroxy;    X=trifluoromethyl; Y=H].

In compounds 20-23 and 29, Z is S. In compounds 24-28, Z is O.

Of the above aldose reductase inhibitors, compound 4 (zenarestat) isespecially preferred.

Said compounds of formula B are prepared as disclosed in U.S. Pat. No.4,939,140.

The aldose reductase inhibitor compounds of this invention are readilyavailable or can be easily synthesized by those skilled in the art usingconventional methods of organic synthesis, particularly in view of thepertinent patent specifications.

The invention also extends to:

-   the use of the said association of the ingredients i) and ii) as    mentioned above in the manufacture of a medicament having    anti-diabetic activity;-   the method of treating or preventing diabetes which comprises    administering to a human or animal an effective dosage of the said    association; and-   kits or single packages combining the active ingredients (i)    and (ii) as mentioned above, useful in treating or preventing    diabetes.    The ingredients i) and ii) of the association can be administered    simultaneously, separately, or sequentially in any order.

Preferably, the invention extends to a method of lowering or maintainingthe glucose blood level by administering to a human or animal aneffective dosage of an extract, or a compound as described above, or acomposition containing the same.

Preferably, the invention extends to a method of lowering or maintainingthe glucose blood level by ingesting a foodstuff or beverage containingan extract, or a compound as described above.

More preferably, the invention also extends to the treatment of impairedglucose tolerance.

Still more preferably, the invention provides a protective effect, inthat the glucose blood level may not substantially increase after thearrest of the administration of an extract, compound, composition and/orfoodstuff or beverage described above.

A method has been described in WO 98/46243 for extracting steroidalglycosides from plant material obtained from a plant of the Trichocaulonor Hoodia genus.

The extract having anti-diabetic activity according to the invention maybe prepared in accordance with the process described in WO 98/46243 forpreparing an extract of a plant of the genus Trichocaulon or of thegenus Hoodia, the extract comprising an appetite suppressant agent.

As described in WO 98/46243 and incorporated herein by reference, theprocess for preparing an extract of a plant of the genus Trichocaulon orof the genus Hoodia comprising a anti-diabetic agent includes the stepsof treating collected plant material with a solvent to extract afraction having anti-diabetic activity, separating the extractionsolution from the rest of the plant material, removing the solvent fromthe extraction solution and recovering the extract. The extract sorecovered may be further purified, e.g. by way of suitable solventextraction procedures.

The extract may be prepared from plant material such as the stems androots of said plants of the genus Trichocaulon or of the genus Hoodia.The genus Trichocaulon and the genus Hoodia include succulent plantsgrowing in arid regions such as are found in Southern Africa. In oneapplication of the invention, the anti-diabetic extract is obtained fromthe species Trichocaulon piliferum. The species Trichocaulon officinalemay also be used to provide an active anti-diabetic extract. In anotherapplication of the invention, the active anti-diabetic extract may beobtained from the species Hoodia curroril, Hoodia gordonii or Hoodialugardii.

The plant material may be homogenised in the presence of a suitablesolvent, for example, a methanol/methylene chloride solvent, by means ofa device such as a Waring blender. The extraction solution may then beseparated from the residual plant material by an appropriate separationprocedure such as, for example, filtration or centrifugation. Thesolvent may be removed by means of a rotary evaporator, preferably in awater bath at a temperature of 60° C. The separated crude extract maythen be further extracted with methylene chloride and water before beingseparated into a methylene chloride extract and a water extract. Themethylene chloride extract may have the solvent removed preferably bymeans of evaporation on a rotary evaporator and the resultant extractmay be further purified by way of a methanol/hexane extraction. Themethanol/hexane extraction product may then be separated to yield amethanol extract and a hexane extract. The methanol extract may beevaporated to remove the solvent in order to yield a partially purifiedactive extract.

The partially purified active extract may be dissolved in methanol, andmay be further fractionated by column chromatography, employing silicagel as an adsorption medium and a chloroform/30% methanol mixture as aneluent. A plurality of different fractions may be obtained, and each maybe evaluated, by suitable bioassaying procedures, to determine theanti-diabetic activity thereof.

A fraction having anti-diabetic activity may preferably be furtherfractionated such as by column chromatography using silica gel as anadsorption medium and a 9:1 chloroform:methanol solvent, and theresultant sub-fractions bioassayed for their anti-diabetic activity. Asub-fraction displaying anti-diabetic activity may, if desired, befurther fractionated and purified, conveniently using a columnchromatographic procedure with silica gel as the adsorption medium and a9:1 ethylacetate:hexane solvent. The resultant purified fractions mayagain be evaluated by suitable bioassay procedures for theiranti-diabetic activity.

The Applicant has found that at least one such purified fraction hasgood anti-diabetic activity, and the active principle in the fractionwas identified by conventional chemical techniques including nuclearmagnetic resonance, and was found to be a compound of the structuralformula (1) as shown above.

According to another aspect of the invention, there is provided aprocess for preparing an extract of a plant of the genus Trichocaulon orof the genus Hoodia, the extract comprising an anti-diabetic agent, theprocess including the steps of pressing collected plant material toseparate sap from solid plant material and recovering the sap free ofthe solid plant material to form the extract.

The extract may be dried to remove moisture, e.g. by spray-drying,freeze-drying or vacuum drying, to form a free-flowing powder.

The steroidal glycosides derivatives of general formula (A) as describedabove having anti-diabetic activity according to the invention may beprepared as described in WO 98/46243.

The molecules chosen as the analogues or derivatives are intended toaffect the properties of the steroidal trisaccharide with the aim ofincreasing the activity of the active ingredient. The following effectswere taken into consideration when the analogues were chosen:

-   (i) Hydrophobic interactions and lipophilicity Functional group    modifications of the active molecule is intended to change the    hydrophobicity and lipophilicity of the molecule. Increased    lipophilicity has been shown to correlate with increased biological    activity, poorer aqueous solubility, increased detergency/cell    lysis, increased storage in tissues, more rapid metabolism and    elimination, increased plasma protein binding and faster rate of    onset of action.-   (ii) Electronic properties and ionization constants Functional group    modification of the molecule is also intended to change the acidity    and basicity which would have a major role in controlling the    transport of the compound to its site of action and the binding at    this target site.-   (iii) Hydrogen bonding Functional group modifications of carboxyl    and carbonyl groups in the active molecule are intended to change    the interactions between the proteins in biological systems and the    chemically modified functional groups.-   (iv) Steric parameters The purpose of changing the steric features    of the molecule is to increase binding to its receptor and thus    increase its biological activity.

The following are examples of the analogues and derivatives inaccordance with this invention:

-   a) Chemical modification of the C-12 group and ester functionality;-   b) Chemical modification of the 5,6-double bond, e.g. hydrogenation    and migration;-   c) Chemical modification of the C-20 carbonyl and C-17 acetyl group;-   d) Chemical modification of the “D” ring of the steroid or aglycone    ring;-   e) Modification of the carbohydrates of the trisaccharide moiety.

Accordingly, the invention provides the compounds of general formula(A), (1), (2), (3), (4), (5), (6), (7), (8), (9), (10), (11), (12),(13), (14) as shown above, wherein in the general formula (A), (2), (3),(4), (5), (6), (7), (8), (9), (10), (11), (12), (13):

R=alkyl;

R₁=H, alkyl, tigloyl, benzoyl, or any other organic ester group;

R₂=H, or one or more 6-deoxy carbohydrates, or one or more 2,6-dideoxycarbohydrates, or glucose molecules, or combinations thereof;

and the broken lines indicate the optional presence of a further bondbetween C4-C5 or C5-C6, and/or C14-C15;

R₃=H, alkyl, aryl, acyl, or glucoxy.

And in the general formula (14):

R=H, alkyl, aryl or any steroid possessing a C14 beta hydroxy group, ora C12 beta hydroxy functionality, or a C17 acyl group, or a C5 - C6olefin, or combinations thereof.

The invention still further extends to a process for syntheticallyproducing a compound having anti-diabetic activity, such as those ofgeneral formula (A), (1), (2), (3), (4), (5), (6), (7), (8), (9), (10),(11), (12), (13), (14).

The process for preparing the compounds of general formula (1), (2),(3), (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), theirintermediates and process for preparing them are described in WO98/46243 and are incorporated herein by reference. Compounds of formula(A) can be prepared by analogy or adaptation of this process.

The invention and its efficacy is further described, without limitationof the scope of the invention, with reference to the examples 1-41 andthe drawings of the application WO 98/46243 and incorporated herein byreference, together with the following examples and drawings.

In the drawings, FIG. 1 shows a flow diagram of the general method ofextracting a first crude anti-diabetic extract and a purifiedanti-diabetic extract from plant material of the genus Trichocaulon orHoodia;

FIGS. 2 and 3 together show a schematic representation of a preferredembodiment of the process of the invention for producing an extract ofplant material of the genus Trichocaulon or Hoodia.

EXAMPLE 1

The effects of compounds of this invention on the glucose and insulinstatus were assessed as follows:

Animals and Husbandry

The animals used for this study were 30 male ZDF rats and 6 male leanZDF rats obtained from Gmi (Indianapolis, Ind., USA). The rats arrivedat 6 weeks of age. The acute study was undertaken when the rats were 7weeks old and the chronic study started when the rats were 9 weeks old.

Animals were housed under the following conditions: Temperature: 23° C.± 1° C. Light: 12 hours light/12 hours dark, lights on at 7 AMAnimals were housed in plastic cages with bedding. Animals were fed astandard laboratory diet (rat and mouse breeding diet, (Beekay Feed, B &K Universal Ltd, Hull, UK)) and drinking water was provided ad libitum.

Experimental Design

Acute Study

This was a single dose, dose-response study in ZDF rats. The 30 ZDF ratswere allocated to one of 5 groups so that there were 6 rats in eachgroup. In addition there were 6 untreated lean ZDF rats. All of the ratswere housed individually. The acute dose of the compound as given byoral gavage at 9.30 AM. Control ZDF and lean rats received water. Foodintake was measured over the periods 9.30-16.00 (daytime) and 16.00-9.30(night-time) for 48 hours.

Chronic Study

After a wash-out of 9 days after the acute dose, the rats were retainedin the same treatment groups for the chronic study. For this study,there were two treatment groups and each treatment group had a pair-fedcontrol group. For the pair-feeding, rats were individually matched.Pair-fed rats together with controls were dosed with water daily. Ratswere dosed daily at ca 9.30 AM. The initial doses were 120 mg/kg (highdose) and 60 mg/kg (low dose).

Food and water intake were measured daily. Bodyweights were measuredtwice weekly. Blood samples were taken for determination of glucose,insulin and leptin. Oral glucose tolerance was also measured.

Acclimatisation

Six days prior to the single dose administration the rats were allocatedto individual cages and were provided with food and water ad libitum.Four days prior to dose administration, 50 g of food was placed in eachcage. Then two days prior to dose administration at ca 9.30 and 16.00hours the food remaining was weighed and replaced with a further 50 g.

Group Allocation

Groups were assigned as follows: Acute Study Group Cage Treatment Doselevel A 1-6 Water 1 ml/kg B  6-12 The compound (1) 20 mg/kg C 13-18 Thecompound (1) 40 mg/kg D 19-24 The compound (1) 80 mg/kg E 25-30 Thecompound (1) 160 mg/kg F 31-36 No treatment (lean litter mates) N/AN/A = Not applicable

Chronic Study Group Cage Treatment Dose level A 1-6 Water 1 ml/kg B 6-12 The compound (1) 120 mg/kg C 13-18 Water (pair fed group B) 1ml/kg D 19-24 The compound (1) 60 mg/kg E 25-30 Water (pair fed group D)1 ml/kg F 31-36 No treatment (lean litter mates) N/AN/A = Not applicableGroups C and D were pair fed, they received the exact amount of food(plus 1 g for waste) eaten by the respective pair rats from the treatedgroups in the previous 24 hours.

Test Compound Administration

Acute Study—The compound (1) was made up in water. Animals wereadministered with a single oral dose of The compound (1) at theappropriate rate. Control animals received water alone at the rate of 1ml/kg. The compound (1) was administered at ca 9.30 AM.

Chronic Study—The compound (1) was made up in water. Animals were dosedat the appropriate rate daily by oral gavage. Control animals receivedwater alone at the rate of 1 ml/kg. Doses were administered at ca 10 AMeach day for a total of 30 days. On Day 7 of the dosing procedure thedose levels of groups B & D were reduced to 60 mg/kg and 30 mg/kg due toconcerns over that food intake suppression might be too great. Thedosing procedure remained the same and animals were dosed for a further23 days (30 days dosing in total).

Experimental Procedures

Measurement of Food and Water Intake

Daily food and water intake of each rat was measured by weight. Anyspilled food was also collected and weighed so that an accurate estimateof food consumption could be made.

Measurement of Bodyweight

The bodyweight of each rat was measured twice weekly throughout thecourse of the study.

Blood Sampling

For measurement of blood glucose levels, a 20 μl sample of blood wastaken from the tail vein whilst animals were in a fed state. A further100 μl sample of blood was taken and the plasma separated bycentrifugation (5000 rpm, 5 minutes). Plasma samples were then analysedfor insulin and leptin using rat insulin or leptin ELISA kits (CrystalChem Inc, PO Box 60225, Chicago, Ill. 60660, USA). Blood glucose,insulin and leptin levels were determined at approximately weeklyintervals.

Fasted glucose, insulin and leptin levels were determined in anidentical manner but animals were fasted for 5 hours prior to samplesbeing taken.

Oral Glucose Tolerance Test (OGTT)

Oral glucose tolerance was measured at 9 days following treatment(chronic study). Animals were fasted for 5 hours prior to the start ofthe OGTT. Animals were treated with glucose diluted in water at a rateof 2 g/kg (1 mg/ml). Blood samples were taken at 0, 30, 60 90 and 120minutes. Glucose concentrations were determined by mixing blood sampleswith 0.38 ml of haemolysis reagent. A duplicate 20 μl aliquots of thismixture was taken for each individual sample and placed in an assaytray. To each well was added 180 μl aliquots of Trinders glucosereagent. The samples were mixed and then left for approximately 30minutes. Samples were then analysed automatically using a SpectraMax 250and SoftMax Pro software (Molecular devices Corporation, 1311 OrleansDrive, Sunnyvale, Calif. 94089, USA). The results were converted intoglucose concentration values using Prism software, version 3.0 (GraphPadSoftware Inc, San Diego, Calif. USA)

The OGTT was repeated after 30 days treatment. The procedure wasidentical except that the pair-fed rats were each given 6 g of food at 7AM with food being withdrawn at 9 AM for 5 hours.

Blood samples for insulin were obtained at 30 minutes prior to and 60minutes post glucose load.

Regression Study

In order to determine the potential long-term effects of The compound(1), the measurement of food and water intake were continued followingwithdrawal of treatment.

Termination of Experiment

The experiment was terminated 36 days after the end of compoundadministration in the chronic study.

Statistical Analysis and Data Compilation

The significance of any differences between control animals and animalstreated with The compound (1) was determined using ANOVA tests.

Results

The values reported in this section are mean values for each group ofanimals. Data for individual animals in are shown in the appendices.

Acute Study

Effect of The compound (1) on Food Intake

The compound (1), over the dose range 20-160 mg/kg, had no significanteffect on daytime food intake over the 7 hour period post-dosing.However, it produced a dose-related reduction in the night-time foodintake such that the food intake such that the food intake of ZDF ratsgiven the 160 mg/kg dose level was the same as the lean rats.

Food intake for the period 24-48 hours past a simple oral dose was alsoreduced (both daytime and night-time) in a dose-related manner.

Significant reduction in food intake was only demonstrated in the first24 hours in rats given The compound (1) (160 mg/kg) but in the secondday both the effects of the 80 and 160 mg/kg doses were significant.

Chronic Study

Effect of the Compound (1) on Food Intake

Daytime and night-time food intake were determined separately. Daytimeintake in untreated ad-lib fed rats was approximately 25% of thenight-time intake. ZDF rats ate more than the lean control rats duringboth day and night-time.

Treatment with The compound (1) at 120 mg/kg and 60 mg/kg produced anincreasing effect on daytime intake over the first 7 days. However thefull effect on night-time intake was apparent after 2 days.

As the reduction in food intake after 7 days was around 50% with bothdose levels, it was decided to reduce the dosing levels to 60 mg/kg and30 mg/kg respectively. Treatment continued for a further 21 days.

Over the period 7-14 days, there was a small reduction in theeffectiveness of The compound (1) on food intake reduction but the rateof change in efficacy after reducing the dose level was slow. After 14days food intake in both groups stabilised.

After withdrawal of the drug treatment after 28 days, food intake wasmonitored for a further 17 days. Surprisingly there was only a smallloss of efficacy relative to the control ZDF rats.

The pattern of feeding in the pair-fed rats was not significantlydifferent from treated animals showing the successful adoption of thepair-feeding regime.

Effect of the Compound (1) on Water Intake

The ZDF rats were 9 weeks old at the start of the chronic study andtheir water intake relative to lean controls indicates that they werediabetic. Thus their intake was around 80 ml/rat/day as against 25ml/rat/day for lean controls. [One control ZDF rat remained non-diabeticthroughout the course of the study and all data from this rat waseliminated from the results].

Treatment with the Compound (1) at either the high dose (120 or 60mg/kg/day) or the low dose (60 or 30 mg/kg/day) reduced water intakewithin 4 days to the level in the lean controls and was maintained atthis level throughout the dosing period. Water intake in the pair-fedgroups was also reduced in an identical manner to the treated groups.

After withdrawal of the treatment after 30 days water intake of bothpreviously treated and pair-fed controls rose slightly but even after 66days the rats were not as diabetic as the untreated controls.

The water intake in the untreated controls rose steadily from theinitial 80 ml/rat/day at the beginning of the study (rats were then 9weeks old) to around 200 ml/rat/day after a further 4 weeks.

Effect of the Compound (1) on Bodyweight

At the start of the chronic study the bodyweight of the ZDF rats wasapproximately 280 g whereas the lean littermates were approximately 220g.

The bodyweight of the untreated lean littermates increased steadilythroughout the course of the study to approximately 380 g at the end ofthe experiment. This contrasted with the control ZDF rats whosebodyweight plateaued after 3 weeks (at the age 12 weeks) at between360-370 g. This plateau in growth rate is presumably due to the severediabetes. Treatment with The compound (1) produced a dose-relateddecrease in the growth rate over the first 3 weeks of treatment relativeto ZDF controls but whereas the growth rate of the ZDF controlsplateaued, the growth of rats given The compound (1) continued andplateaued at a much higher level (more than 400 g). The growth rate ofthe pair-fed rats mirrored the effect of their corresponding treatedgroups. There was actual actual gain of bodyweight for the ZDF rat overthe treatment period.

Effect of the Compound (1) on Glucose Concentration in Fed Rats

Treatment with The compound (1) and pair feeding to the intake of ZDFrats given The compound (1) resulted in a reduction in the blood glucoseconcentration from the diabetic level to a similar concentration as inlean littermates after 7 days of treatment.

Normal glycaemia was maintained until withdrawal of therapy when bloodglucose concentration steadily increased in a similar manner in bothrats previously given The compound (1) and their pair-fed controls.

Effect of the Compound (1) on Blood Glucose Concentration in Fasted Rats

Animals were fasted for 5 hours prior to taking a blood sample. Bothafter 8 days treatment and 29 days treatment, the blood glucoseconcentration of rats given The compound (1) and their pair-fed controlsdid not differ significantly from the concentration in lean rats and wassignificantly lower than that in ZDF controls.

Effect of the Compound (1) on Oral Glucose Tolerance

Oral glucose tolerance was determined after 8 days and after 29 days oftreatment, i.e. on day 9 and day 30. Rats were fasted for 5 hours priorto receiving an oral 2 g/kg glucose load.

The fasting blood glucose in the ZDF control rats on day 9 was around 11mM and after the oral glucose load, rose to a mean of more than 14 mM.In contrast, rats given The compound (1) and their pair-fed controls hadfasting blood glucose concentrations similar to the lean rats andglucose tolerance was only marginally impaired relative to lean. Similarresults were obtained in the study conducted on day 29 except that thefasting blood glucose of the ZDF rats was higher, indicative of theiradvancing diabetic state.

DISCUSSION

The inbred (>F₃₀ generations) Zucker diabetic male fatty rat is arecently developed model of non-insulin dependent diabetes. It is on theZucker background and the fa gene. The original obesity trait in Zuckerrats was identified by Zucker and Zucker and has since been maintainedin numerous locations around the world. The original non-inbred ratmodel is associated with massive obesity, hyperinsulinaemia and glucoseintolerance but not frank diabetes.

In contrast, the inbred ZDF/Gmi, which had its origin in a non-inbredcolony in which some obese rats developed overt diabetes (1),demonstrates a characteristic and consistent diabetes (2, 3).Hyperglycaemia is initially manifest at about 7 weeks of age and allobese males are fully diabetic by 10 weeks with fed blood glucoseconcentrations of about 30 mM. Between 7 and 10 weeks, blood insulinconcentrations fall as the pancreatic β-cells cease to respond to theglucose stimulus (4-9). The loss of response to glucose appears to beassociated with the disappearance of GLUT-2 transporters on the β-cellsin the islets. There is also a reduced number of GLUC-4 transporters inskeletal muscle (10-12). Thus the ZDF rat shows both an impairment ofinsulin action, i.e. insulin resistance and an insulin secretory defectand is recognised as a good model of non-insulin dependent diabetes. Thefirst-line treatment for non-insulin dependent diabetes in man is dietplus exercise. Whilst the original dietary concept was a reduction inintake of carbohydrate, today it is focussed on a weight reducing dietthat is low in fat, contains significant carbohydrate as polysaccharidesbut is low in mono and disaccharides. It should also be high in fibre.If body weight can be reduced by 5 kg then a marked improvement indiabetic control can be achieved. In practice relatively few diabetics(more than 90% of non-insulin dependent diabetic patients areoverweight) are able to achieve and maintain such weight loss. Thus,there is a clear indication for therapeutic agents that will produce areduction in obesity for the treatment of non-insulin dependentdiabetes. Indeed, FDA guidelines for anti-obesity drugs specificallyrecognise treatment of diabetes as a secondary end-point. The compound(1) has previously been shown to reduce food intake in normal rats whenadministered orally. However, no previous studies have been undertakenin obese animals, which might respond differently. Furthermore, energyintake and expenditure are often closely linked and it was possible thatThe compound (1) might exert independent effects on intake andexpenditure. Thus, as a control for possible effects unrelated to foodintake, pair-fed controls were included in the current study. A furtherpotential of the present study was to examine the possible developmentof pharmacological tolerance.

One previous study (13) in pre-diabetic ZDF rats has demonstrated thatif 6 week old ZDF rats were diet-matched with lean littermates for 12weeks, they remained euglycaemic. Since the rats at 6 weeks of age arepre-diabetic, then this merely demonstrates that dietary restrictionwill prevent the development of diabetes in this model. The presentstudy is the first to examine the treatment of established diabetes. Inorder to establish dose levels for the chronic study, a single dose,dose-response study was undertaken to examine the effect on food intake.Surprisingly the effect of The compound (1) appeared to be slow in onsetwith little apparent effect over the first few hours. However, since thedose was administered at 09.30 and daytime food intake is onlyapproximately 20% of the 24 h intake, it is not absolutely clear thatthere is a time-delay in the response, but it appears likely. Furtherstudies in which the dose is administered just before the dark-phase areneeded to clarify this point.

The duration of response to a single dose was long without effects stillbeing seen during the second day after a single dose. As a result of thesingle dose study doses of 120 and 60 mg/kg/day were selected for thechronic study.

The chronic administration of these dose levels produced a somewhatgreater effect on food intake than was expected, possibly because ofslow elimination of The compound (1). To avoid possible adverse effectsof severe calorie restriction, the dose-levels were reduced after 7 daysto 60 and 30 mg/kg respectively.

The compound (1) produced a marked reduction in food intake, which wassustained with no indication of tolerance. This reduction in food intakewas reflected in lower initial growth rates. However, after 14 days,rats given The compound (1) had a higher growth rate than the controlZDF rats. This was because the ZDF controls had reached a plateauweight, presumably as a result of their severe diabetes. By the end ofthe treatment period the ZDF rats given the low dose of The compound (1)were actually heavier than the controls whilst the high dose Thecompound (1) rats were the same weight.

Withdrawal of the drug surprisingly did not lead to a reboundhyperphagia. Whether this indicates a long wash-out period for Thecompound (1) or it is a reflection of the difference in the diabeticstatus of the control ZDF rats and rats given The compound (1) is notknown.

In parallel with the changes in food intake, there were substantialimprovements in glycaemic status. This was reflected in directmeasurements of plasma glucose and glucose tolerance as well as waterintake. Since the ZDF control rats exhibit glycosuria they areprofoundly polydipsic. Reduction in glycosuria results in substantialreduction in water intake. Thus the daily water intake gives an indirectmeasure of the level of diabetic control. It is clear that dietaryrestriction of ZDF rats whether direct or through the use of Thecompound (1) results in substantial improvement in glycaemic control.

Withdrawal of the treatment did not result in an immediate return to thediabetic state. In fact, it was approximately 2 weeks before the bloodglucose concentration and water intake of rats previously given Thecompound (1) approached that of the diabetic controls. By which time thebody weight of the previously treated rats was significantly greaterthan that of the ZDF controls.

The Gmi ZDF rats retain the fa/fa gene which result in point mutation inthe extracellular domain in the leptin receptor conferring insensitivityto leptin. Thus the ZDF rats are obese but the obesity is curtailed bythe diabetic condition. The obesity via its action on insulinsensitivity contributes to the diabetic status but the major defect inthese rats that results in diabetes is pancreatic. This defect must beunrelated to the fa/fa mutation. It is clear from the above studies thatThe compound (1) acts on food intake independently of leptin.

CONCLUSION

The compound (1) is a powerful appetite suppressant in ZDF male rats andis an effective treatment in treating established diabetes.

REFERENCES

-   1. Clark, J. B., Palmer, C. J. & Shaw, W. N. (1983). The diabetic    Zucker fatty rat. Proc. Soc. Exp. Biol. Med., 173, 68-75.-   2. Peterson, Richard G. (1995). The Zucker Diabetic Fatty (ZDF) rat.    In: Shafrir, E. ed. Lessons from Animal Diabetes V. Great Britain,    Smith-Gordon, 225-230.-   3. Peterson, R. G. (1994). Alfa Glucosidase Inhibitors: Lessons from    Animal Studies. European Journal of Clinical Investigation, 24(S3),    11-18.-   4. Johnson, J. H., Ogawa, A., Chen, L., Orci, L., Newgard, C. B.,    Alam, T. & Unger, R. H. (1990). Underexpression of b cell high K_(M)    glucose transporters in non-insulin dependent diabetes. Science,    250, 546-549.-   5. Lee, Y., Hirose, H., Ohneda, M., Johnson, J. H., McGarry, J. D. &    Unger, R. H. (1994). Beta-cell lipotoxicity in the pathogenesis of    non-insulin dependent diabetes mellitus of obese rats: impairment in    adipocyte-beta-cell relationships. Proc. Natl. Acad. Sci. USA,    91(23), 10878-10882.-   6. Milburn, J. L. Jr., Ohneda, M., Johnson, J. H. & Unger, R. H.    (1993). Beta-cell GLUT-2 loss and non-insulin dependent diabetes    mellitus: current status of the hypothesis. (Review) Diabees Metab.    Rev., 9(3), 231-236.-   7. Pieber, T. R., Stein, D. T., Ogawa, A., Alam, T., Ohneda, M.,    McCorkle, K., Chen, L., McGarry, J. D. & Unger, R. H. (1993).    Amylin-insulin relationships in insulin resistance with and without    diabetic hyperglcemia. Am. J. Physiol., 265(3 Pt. 1), E446-E453.-   8. Slieker, L. J., Sundell, K. L., Heath, W. F., Osborn, H. E., Bue,    J., Manetta, J. & Sportsman, J. R. (1992). Glucose transporter    levels in tissues of spontaneously diabetic Zucker fa/fa rat    (ZDF/Drt) and viable yellow mouse (A^(vy)/a). Diabetes, 41, 187-193.-   9. Sturis, J., Pugh, W. L., Tang, J., Ostrega, D. M.,    Polonsky, J. S. & Polonsky, K. S. (1994). Alterations in pulsitile    insulin secretion in the Zucker diabetic fatty rat. Am. J. Physiol.,    267 Endocrinol. Metab., 30, E250-259.-   10. Dohm, G. L., Friedman, J. E. & Peterson, R. G. (1993). Acarbose    treatment of non-insulin dependent diabetic fatty (ZDF/Drt-fa) rats    restores expression of skeletal muscle glucose transporter GLUT-4.    In: Drugs in Development, Volume 1, a-Glucosidase Inhibition:    Potential Use in Diabetes. Vasselli, J. R., Maggio, C. A. &    Scirabine, A., eds., Branford, Ct.: Neva Press, 173-180.-   11. Dolan, P. L., Tapscott, E. B., Peterson, R. G. & Dohm, L. D.    Differential effects of acarbose feeding on glucose transport and    GLUT-4 protein in lean and obese diabetic ZDF rats. Submitted for    publication.-   12. Friedman, J. E., De Vente, J. E., Peterson, R. G. & Dohm, G. L.    (1991). Altered expression of muscle glucose transporter GLUT-4 in    diabetic fatty Zucker rats (ZDF/Drt-fa). Am. J. Physiol., 261    (Endocrinol. Metab. 24), E782-788.-   13. Ohneda, M., Inman, L. R. & Unger, R. H. (1995). Caloric    restriction in obese pre-diabetic rats prevents beta-cell depletion,    loss of beta-cell GLUT-2 and glucose incompetence. Diabetologia,    38(2), 173-179.

EXAMPLE 2

Harvested Hoodia plants received either from the natural environment orthrough a cultivation programme are first stored at 4° C. for a maximumof 48 hours. The plants are washed in tap water and thereafter slicedinto ±1 cm slices. The sliced pieces are all combined and then pressedthrough a hydraulic press at 300 bar pressure for a minimum of 0.5 hourper pressing. During the pressing the sap of the plant is collectedseparately. The sap is stored at −18° C. until further processing isrequired.

The sap is spray-dried under suitable conditions to obtain a freeflowing powder. The moisture content in the powder is preferably lessthan 5% after spray drying and, if necessary, it is further dried in avacuum oven or using a fluid bed drier.

Both the sap and the spray-dried material have been shown effective asan anti-diabetic in biological assays in rats.

Experimental

50 kg of Hoodia gordonii plants were washed with tap water andthereafter sliced into 1 cm slices. The sliced plants were then pressedthrough a hydraulic press at 300 bar for a minimum of 0.5 hour perbatch. The sap was collected and the mass was found to be 10 kg whenHoodia gordonii plants from the environment were used, and 20 kg whenHoodia gordonii plants from the cultivation programme was used. The sap(500 g) was spray-dried using the following conditions: Flow rate 2.85ml/min Inlet temperature 110° C. Outlet temperature  70° C. Chambertemperature  78° C.

The spray-dried powder obtained was a free flowing powder (22 g) with amoisture content of 6.9%.

The spray dried powder was analysed for active ingredient concentrationusing HPLC techniques. The concentration of the active was determined tobe 13 g/kg of spray dried powder.

HPLC Analysis Method Eluant Acetonitrile: water (7:3), isocratic ColumnReverse phase C-18 UV absorbance 225 nm Flow rate 1 ml/min Injectionvolume 10 μl

Method

Spray-dried powder (10 mg) was dissolved in water (0.5 ml) andacetonitrile (0.5 ml) 10 μl of this solution was injected into the HPLCand the concentration of the active compound (1) was determined using astandard curve as a reference which had been prepared from the purecompound (1).

It should be understood that the foregoing discussion and examplesmerely present a detailed description of certain preferred embodiments.It will be apparent to those of ordinary skill in the art that variousmodifications and equivalents can be made without departing from thespirit and scope of the invention. All the patents, journal articles andother documents discussed or cited above are herein incorporated byreference.

1. A method of treating or preventing diabetes by administering to ahuman or animal an effective dosage of an extract of a plant of thegenus Trichocaulon or of the genus Hoodia. 2-34. (canceled)